Compositions and Methods for Inhibiting Pro-Inflammatory Cytokine Gene Expression

ABSTRACT

θ-defensins, non-human macrocyclic peptides, have been found to reduce expression of genes encoding for various pro-inflammatory peptides (such as cytokines and chemokines) in a highly selective manner. Methods for treating inflammatory conditions utilizing a θ-defensin and/or a θ-defensin analog, methods for modifying (e.g. down regulating) gene expression for pro-inflammatory peptides in a subject in need thereof using a θ-defensin and/or a θ-defensin analog, methods for selectively modifying expression of such genes and/or reducing pro-inflammatory peptides in a subject without inducing or worsening immunosuppression using a θ-defensin and/or a θ-defensin analog are discussed, as are compositions that include a θ-defensin and/or a θ-defensin analog in an amount and/or form suitable for use in such methods.

This application claims priority to U.S. Provisional Application No.62/202,403 filed on Aug. 7, 2015. These and all other referencedextrinsic materials are incorporated herein by reference in theirentirety. Where a definition or use of a term in a reference that isincorporated by reference is inconsistent or contrary to the definitionof that term provided herein, the definition of that term providedherein is deemed to be controlling.

This invention was made with government support under Grant No. AI022931awarded by the National Institute of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The field of the invention is methods and compositions effective inreducing the effect of pro-inflammatory cytokines, specificallyinhibiting expression of cytokine genes.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Inflammation is a complex protective response to pathogens, tissuedamage, and exposure to irritants, involving alteration in bloodvessels, mobilization of immune cells, and release of a variety ofchemical and peptide mediators. While inflammation serves to remove theinitial cause of cell injury and eliminate necrotic cells from damagedtissue, the inflammatory response can itself be damaging. For example,chronic inflammation resulting from autoimmune disease can contribute todamage of the affected tissue. Similarly, inflammation resulting fromacute processes, such as viral or bacterial infection, can result intissue damage and septic shock. In addition the pain and swelling thataccompany inflammation can be debilitating, particularly when it is theresult of chronic conditions.

Unfortunately, current methods for treating inflammation suffer from anumber of drawbacks. For example, traditional pharmaceutical approaches(e.g. treatment with steroids or non-steroidal inflammatory drugs)provide only short term relief, and often do so at the cost ofsignificant side effects that limit the use of such drugs. Morerecently, “biologics” (for example, humanized monoclonal antibodies toproinflammatory cytokines) have been used to treat certain chronicconditions characterized by inflammation, however such approachesnecessarily target only a single inflammation mechanism, and can resultin immune suppression or even an immunocompromised state in a treatedindividual.

Mammalian defensins are cationic, tri-disulfide-containing peptidescomprising three subfamilies denoted as α-, β-, and θ-defensins.Defensins contribute to host defense as antimicrobial agents (Ericksenet at, Antimicrob Agents Chemother 49:269-275 (2005)) and by regulatinginflammatory (Khine et at, Blood 107:2936-2942 (2006)) and adaptiveimmune responses (Chertov et at, J Biol Chem 271:2935-2940 (1996)). Allpublications herein are incorporated by reference to the same extent asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference. Where adefinition or use of a term in an incorporated reference is inconsistentor contrary to the definition of that term provided herein, thedefinition of that term provided herein applies and the definition ofthat term in the reference does not apply. The α- and β-defensins havesimilar three dimensional topologies but differ in their disulfidelinkages (Selsted and Ouellette, Nat Immunol 6:551-557 (2005)).θ-defensins are macrocyclic peptides that are expressed in Old Worldmonkeys (e.g., macaques and baboons) and are the only known cyclicproteins in animals (Lehrer et at, J Biol Chem 287:27014-27019 (2012)).This basic θ-defensin backbone structure is produced by head-to-tailsplicing of two nonapeptides that are excised from α-defensin-likeprecursors (Tang et al, Science 286:498-502 (1999)). In rhesus macaques,alternate binary splicing of nonapeptides encoded by three precursorgenes provides six θ-defensin isoforms, rhesus theta-defensins (RTDs)1-6 (Tang et at, Science 286:498-502 (1999); Leonova et at, J LeukocBiol 70:461-464 (2001)). In baboons, alternate nonapeptide splicingproduces ten θ-defensin isoforms (Garcia et at, Infect Immun76:5883-5891 (2001)). θ-defensins are expressed at high levels ingranules of neutrophils and in monocytes. These θ-defensins play a majorrole in the antimicrobial activities of rhesus neutrophil granuleextracts. The RTD-1 isoform is the most abundant θ-defensin in macaques,constituting approximately 55% of the total θ-defensin content of rhesusneutrophils (Tongaonkar et al, J Leukoc Biol 89:283-290 (2011)).

Humans and other hominids lack θ-defensins due to the presence of a stopcodon in the prepro-coding sequence of θ-defensin genes in these species(Nguyen et at, Peptides 24:1647-1654 (2003)). It has been suggested thatthe expression of θ-defensins in Old World monkeys is related todifferences in immune and inflammatory responses of these nonhumanprimates from those of humans (Leher et at, J Biol Chem 287:27014-27019(2012)).

While α-, β-, and θ-defensins were initially identified on the basis oftheir broad spectrum antimicrobial properties, subsequent studies havedisclosed immune regulatory roles for these peptides (Yang et al, AnnuRev Immunol 22:181-215 (2004)). For example, some α- and β-defensins arechemotactic for T cells, neutrophils, dendritic cells, and monocytes(Chertov et at, J Biol Chem 271:2935-2940 (1996); Yang et at, Science286:525-528 (1999); Grigat et at, J Immunol 179:3958-3965 (2007); Soruriet at, Eur J Immunol 37:2474-2486 (2007)), and they induce secretion ofproinflammatory cytokines from activated dendritic cells, peripheralblood mononuclear cells and epithelial cells (Khine et at, 107:2936-2942(2006); Boniotto et at, Antimicrob Agents Chemother 50:1433-1441 (2006);Ito et at, Tohoku J Exp Med 227:39-48 (2012); Yin et al, Immunol 11:37.(2010); Niyonsaba et al, J Immunol 175:1776-1784 (2005); Li et at,Invest Ophthalmol Vis Sci 50:644-653 (2009); Syeda et al, J Cell Physiol214:820-827 (2008)). In contrast to these pro-inflammatory activities,it has recently been reported that θ-defensins have anti-inflammatoryproperties both in vitro and in vivo. For example, RTD-1 was found to bea potent inhibitor of cytokine secretion by human peripheral bloodleukocytes stimulated with diverse Toll-like receptor (TLR) agonists(Schaal et at, PLoS One 7, e51337 (2012)). Naturally occurringθ-defensin isoforms (RTDs 1-6) possess variable potency in reducing TNFin LPS- or E. coli-stimulated leukocytes. (Schaal et al, PLoS One 7,e51337 (2012)). RTD-1 has also been found to reduce inflammatorycytokines, including TNF-α, IL-1β and several chemokines in mouse modelsof SARS corona virus infection (Wohlford-Lenane et al, J Virol83:11385-11390 (2009), in E. coli peritonitis, and in polymicrobialsepsis (Schaal et at, PLoS One 7, e51337 (2012)). Such responses,however, appear to be restricted to specific cytokines, and themechanism of action is not clear.

Thus, there is still a need for methods and compositions that providerelief of inflammation through suppression of a range or plurality ofpro-inflammatory moderators.

SUMMARY OF THE INVENTION

The inventive subject matter provides apparatus, systems and methods inwhich a θ-defensin and/or a θ-defensin analog is provide in amounts andin a form that provides suppression of the expression of one or moregenes encoding for a pro-inflammatory peptide(s), such as a cytokineand/or chemokine. Suitable θ-defensins include RTD-1 (SEQ ID NO. 1),RTD-2 (SEQ ID NO. 6), RTD-4 (SEQ ID NO. 7), RTD-5 (SEQ ID NO. 8), andRTD-6 (SEQ ID NO. 9). Embodiments of the inventive concept includemethods for treating inflammatory conditions utilizing a θ-defensinand/or a θ-defensin analog, methods for modifying (e.g. down regulating)gene expression for pro-inflammatory peptides in a subject in needthereof using a θ-defensin and/or a θ-defensin analog, methods forselectively modifying expression of such genes and/or reducingpro-inflammatory peptides in a subject without inducing or worseningimmunosuppression using a θ-defensin and/or a θ-defensin analog, andcompositions that include a θ-defensin and/or a θ-defensin analog in anamount and/or form suitable for use in such methods.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts typical data demonstrating that RTD-1 inhibits secretionof pro-inflammatory cytokines. TNF-α, IL-1β and IL-8 secreted in themedium were quantified by ELISA. Left panels. Medium from humanmonocytes stimulated for 4 hours with 20 ng/ml LPS or from unstimulatedcells +/−RTD-1 (10 μg/ml). Results are from one of two similar,independent experiments. Right panels. Medium from differentiated THP-1cells treated with LPS (100 ng/ml) for 2 hours or from untreated cells+/−RTD-1 (10 μg/ml). Results shown are mean+/−SD from 3 independentexperiments. # indicates cytokine was below detection limit.

FIG. 2 depicts typical data demonstrating that RTD-1 down regulates mRNAof pro-inflammatory cytokines. Real time PCR analysis of cDNA fromdifferentiated THP-1 cells treated with LPS (100 ng/ml)+/−RTD-1 (10μg/ml) for 2 hours or from control untreated cells. Results shown aremean+/−SD from 3 independent experiments.

FIGS. 3A to 3E depict typical data demonstrating that RTD-1 inhibitsNF-κB activation in cells stimulated with TLR agonists. SEAP activity inthe medium of cells stimulated with TLR agonists in presence or absenceof RTD-1 was assayed using Quantiblue. SEAP activity is expressed asfold induction compared with untreated control cells. In FIG. 3Adifferentiated THP-1 Dual cells were treated with or without LPS (100ng/ml) in the presence or absence of 10 μg/ml RTD-1. In FIG. 3B cellswere treated with or without LPS (100 ng/ml) and in the presence orabsence of either RTD-1 or HNP-1 (numbers indicate concentrations ofRTD-1 and HNP-1 in μg/ml; 2.4 and 4.8 μM correspond to 5 and 10 μg/mlRTD-1 and 8.25 and 16.5 μg/ml HNP-1 respectively). In FIG. 3C the upperpanel shows the structures of RTD-1 and S7 peptide where the dottedlines indicate disulfide linkage, whereas the lower panel shows datafrom THP-1 Dual cells stimulated with 100 ng/ml LPS in the presence orabsence of 10 μg/ml RTD-1 or the acyclic S7 peptide. FIG. 3D showsresults from differentiated THP-1 Dual cells treated with Pam3CSK4 (25ng/ml) in the presence or absence of 10 μg/ml RTD-1. FIG. 3E showsresults from HEK-Blue hTLR9 cells treated with or without ODN2006 (ODN)and +/−10 μg/ml RTD-1. The numbers indicate the concentration of ODN2006in μg/ml. Results are shown as means+/−SD from 3 independentexperiments. * P<0.05 when compared to treatment with agonist alone.

FIGS. 4A and 4B depict typical data demonstrating that Inhibition ofNF-κB DNA-binding activity by RTD-1. Nuclear extracts containing 2.8 μgprotein from control differentiated THP-1 cells and stimulated with (inFIG. 4A) LPS (100 ng/ml), or (in FIG. 4B) TNF-α (2 ng/ml) and with orwithout 10 μg/ml RTD-1 were assayed for DNA-binding. Results aremeans+/−SD from 3 independent experiments. * P<0.05; compared to LPS orTNF-α treated samples. Lower panels of FIG. 4A and FIG. 4B show Westernblots of respective THP-1 nuclear extracts containing 8.1 μg proteinused for DNA-binding assay probed with anti-NF-κB p65 antibodies.

FIGS. 5A and 5B depict typical data demonstrating differential effectsof RTD-1 and the TACE inhibitor marimastat (MM). FIG. 5A shows resultsfrom studies where THP-1 macrophages were treated with or without LPS(100 ng/ml) in the presence or absence of MM and RTD-1. TNF-α (blackbars) and IL-1β (open bars) secreted in the medium were then measured byELISA. Results are means+/−SD from 3 independent experiments. FIG. 5Bshows results from studies where differentiated THP-1 Dual cells werestimulated with 100 ng/ml LPS and co-incubated with the indicatedreagents and NF-κB activity was measured relative to control using theQuantiblue assay. Results shown are means+/−SD from 3 independentexperiments. * P<0.05 when compared to MM+LPS treatment.

FIG. 6 depicts typical data demonstrating that RTD-1 stabilizes IκBα andblocks phosphorylation of p38 MAP and JNK kinases. Cell extracts (15 μgprotein) were obtained from THP-1 cells stimulated with diluent, 100ng/ml LPS, LPS+10 μg/ml RTD-1, or RTD-1 alone. Extracts were resolved onSDS-tricine gels and western blots were probed with anti-IκBα,anti-Phospho p38 MAP kinase and anti-Phospho SAP/JNK kinase antibodies.

FIG. 7 depicts typical data demonstrating that RTD-1 modulatesphosphorylation of multiple inflammatory signaling proteins. Extractsfrom control, 100 ng/ml LPS treated, 100 ng/ml LPS+10 μg/ml RTD-1treated, or 10 μg/ml RTD-1 treated THP-1 cells were used to probe aPhospho-MAPK array. Positive controls were spotted at (A1, A2), (A21,A22) and (F1, F2) and negative control at (E19, E20). Dot intensitiesfrom two independent experiments were quantified with ImageJ software,normalized and the mean values were plotted (lower panel). RTD-1suppressed LPS induced phosphorylation of 7 proteins implicated inLPS-induced inflammatory signaling. Treatment of THP-1 macrophages withpeptide alone increased Akt phosphorylation.

FIGS. 8A to 8C depict typical data demonstrating that RTD-1 stimulationof Akt phosphorylation. In FIG. 8A THP-1 cells were treated with diluentor 10 μg/ml RTD-1 and extracts were prepared from cells harvested at theindicated times. Extracts were resolved on SDS-tricine gels and probedusing anti-Phospho-Akt (P-Akt) and anti-Akt antibodies (upper panel).Band intensities were quantified with ImageJ software, normalizedrelative to total Akt, and the average fold-increase of P-Akt from twoindependent experiments was plotted as function of incubation time(lower panel). In FIG. 8B THP-1 macrophages were incubated with 10 ug/mlof RTD-1 or diluent+/−10 μM Ly294002. Western blots were performed usingAkt or P-Akt antibodies. In FIG. 8C THP-1 Dual cells were pretreatedwith Ly294002 for 60 minutes and then stimulated overnight with 100ng/ml LPS in the presence of 10 μg/ml RTD-1. Medium was analyzed forSEAP activity and fold stimulation was calculated for each Ly294002concentration relative to cells treated with no Ly294002. Results aremean+/−SEM from four independent experiments and the effect of Ly294002treatment on SEAP expression was significant (P<0.05) at allconcentrations tested.

FIG. 9 depicts typical data demonstrating the effect of RTD-1 onsignaling pathways in human monocytes. Extracts (˜7 μg protein) fromunstimulated monocytes or monocytes stimulated with 100 ng/mlLPS+/−RTD-1 (10 μg/ml) for 30 minutes were resolved on SDS-Tricine gelsand the immunoblots were probed with antibodies against IκBα,Phospho-p38 MAP kinase and Phospho-Akt.

FIG. 10 schematically depicts immunoregulatory effects of RTD-1. Theschematic shows the suppression of NF-κB activation stimulated byextracellular (TLR 1/2 & 4) and intracellular (TLR9) receptors,inhibitory and stimulatory effects of RTD-1 on LPS-stimulated pathways,and negative regulatory pathways mediated by P-Akt.

DETAILED DESCRIPTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The inventive subject matter provides compositions and methods in whicha θ-defensin (for example RTD-1 (SEQ ID NO. 1) and/or an RTD-1 analog,derivative, or mutant) is used to suppress expression of genes encodingtwo or more pro-inflammatory moderators. Such pro-inflammatorymoderators can include cytokines and/or chemokines. This genesuppression results in a reduction or elimination of inflammatoryprocesses across multiple inflammatory pathways.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingfigures.

One should appreciate that the disclosed techniques provide manyadvantageous technical effects including suppression of multiplepro-inflammatory mediators with a single compound. In addition, specificsuppression of such pro-inflammatory mediators at the gene expressionlevel is highly selective compared to current treatment modalities andcan result in reduced side effects. Such an approach can also provide anextended duration of the effect of a single dose relative to prior artapproaches,

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Abbreviations and acronyms used throughout this application include:

-   -   BCA Bicinchoninic Acid    -   CCL Chemokine (C-C motif) ligand    -   CLP Cecal ligation and puncture    -   CREB cAMP response Element Binding protein    -   CT Cycle Threshold    -   DMSO Dimethyl Sulfoxide    -   ERK Extracellular signal Regulated Kinase    -   FBS Fetal Bovine Serum    -   gDNA Genomic DNA    -   HI Heat Inactivated    -   HOAc Acetic acid    -   HNP Human neutrophil peptide    -   HSP Heat shock protein    -   IL Interleukin    -   IKK IκB kinase    -   LPS Lipopolysaccharide    -   MAP kinase Mitogen activated protein kinase    -   MM Marimastat    -   NF-κB Nuclear Factor kappa    -   ODN Oligo deoxynucleotide    -   PBS Phosphate buffered saline    -   PCR Polymerase Chain Reaction    -   PI3K Phosphatidyl inositol 3 phosphate kinase    -   PMA Phorbol 12-myristate 13-acetate    -   PMSF Phenyl methyl sulfonyl fluoride    -   PPC Positive PCR control    -   RTC Reverse transcription control    -   RTD Rhesus theta defensin    -   SAPK/JNK Stress activated protein kinase/Jun N-terminal kinase    -   SEAP Secreted embryonic alkaline phosphatase    -   TACE/ADAM17 Tumor necrosis factor alpha converting enzyme/A        disintegrin and metalloproteinase domain 17    -   TAK1 TGF β activated kinase 1    -   TLR Toll like receptor    -   TNF Tumor necrosis factor

Studies on θ-defensins have revealed the pleiotropic properties of thesemacrocyclic host defense molecules. Initially isolated based on theirantibacterial and antifungal properties in vitro, subsequent studieshave demonstrated broader host defense properties mediated by theirantiviral properties against Herpes Simplex virus, HIV and influenza,and arming of phagocytes for enhanced killing of Bacillus anthracis.Protective effects of systemically administered RTD-1 are observed inmouse models of SARS coronavirus infection and sepsis. To gain insightsinto the immunomodulatory mechanisms mediated by θ-defensins,inventor(s) analyzed the effects of RTD-1 on inflammatory pathways inTHP-1 macrophages and have, surprisingly, identified a previouslyunknown mode of action.

The inventor contemplates that properties identified as being associatedwith RTD-1 and/or other θ-defensins are to be found in analogs of thesecyclic peptides. An analog of a θ-defensin can have greater than 70%,75%, 80%, 85%, 90%, 95%, or more amino acid sequence identity to acorresponding parent θ-defensin. Suitable analogs can be based on RTD-1(SEQ ID NO. 1), RTD-2 (SEQ ID NO. 5), RTD-3 (SEQ ID NO. 6), RTD-4 (SEQID NO. 7), RTD-5 (SEQ ID NO. 8), and/or RTD-6 (SEQ ID NO. 9) as parentθ-defensins. Differences in amino acid sequence can be produced bydeletion, addition, and/or substitution of one or more amino acids. Suchsubstitutions can be conservative (i.e. replacement with an amino acidhaving similar charge, size, reactivity, and/or hydrophobicity) ornon-conservative (i.e. replacement with an amino acid having a differentcharge, size, reactivity, and/or hydrophobicity). Such analogs canincorporate non-naturally occurring amino acids, amino acid analogs,and/or amino acid modifications via conjugation (such as PEGylation).Such analogs can be truncated versions of a θ-defensin in which one ormore amino acids of the parent θ-defensin have been deleted, but thatretain essential conformation and structure. For example, the syntheticcyclic peptides designated RTD 1-27 (SEQ ID NO. 2), RTD 1-28 (SEQ ID NO.3), and/or RTD 1-29 (SEQ ID NO. 4) can be considered analogs of RTD-1(SEQ ID NO. 1). Similarly, analogs of RTD-2 (SEQ ID NO. 5), RTD-3 (SEQID NO. 6), RTD-4 (SEQ ID NO. 7), RTD-5 (SEQ ID NO. 8), and RTD-6 (SEQ IDNO. 9) can be used in embodiments of the inventive concept. In someembodiments such a θ-defensin analog can have improved pharmacologicalproperties (e.g. solubility, serum half-life, bioavailability, oral ormucosal absorption, etc.) relative to the related parent θ-defensin.

One embodiment of the inventive concept is a method for treating anacute and/or chronic inflammatory condition by inhibition of the geneexpression of one, two, or more than two pro-inflammatory peptides (e.g.cytokines), where such inhibition is provided by application of aθ-defensin and/or θ-defensin analog to a subject in need ofanti-inflammatory treatment. Dosage and dosing frequency of theθ-defensin and/or θ-defensin analog can be selected to provide relieffrom inflammation and/or measurable reduction in pro-inflammatorypeptide expression in less than 10 minutes, less than 15 minutes, lessthan 20 minutes, less than 30 minutes, less than 1 hour, less than 2hours, less than 3 hours, less than 4 hours, less than 6 hours, lessthan 8 hours, less than 12 hours, less than 16 hours, and/or less than24 hours from administration of an initial dose. Relief frominflammation and/or reduction in pro-inflammatory peptide expression canpersist for at least 1 hour, at least 2 hours, at least 4 hours, atleast 6 hours, at least 8 hours, at least 12 hours, at least 16 hours,at least 24 hours, at least 48 hours, at least 72 hours, and/or morethan 72 hours from establishment of the anti-inflammatory or reductionof pro-inflammatory peptide expression effect. Dosing schedules can beselected for dosing intervals ranging from three times a day to once aweek. In some embodiments the θ-defensin and/or θ-defensin analog can beprovided as a constant infusion. Acute inflammatory conditions that canbe treated in this manner are characterized by sudden and severe onset,and include sepsis, traumatic injury, thermal burns, chemical burns,electrical burns, radiation burns, acute allergic reactions, andinhalation injuries. Chronic inflammatory conditions that can be treatedin this manner are characterized by gradual onset and persistence overtime, and include rheumatoid arthritis, ankylosing spondylitis,inflammatory bowel disease, ulcerative colitis, autistic enterocolitis,psoriasis, psoriatic arthritis, Crohn's disease, Behcet's disease,lupus, hidradenitis suppurativa, refractory asthma, colitis, dermatitis,diverticulitis, hepatitis, nephritis, Parkinson's disease, Alzheimer'sdisease, Huntington's disease, congestive heart disease,atherosclerosis, and uveitis.

Another embodiment of the inventive concept is a method of inhibitinggene expression of one, two, or more than two pro-inflammatory peptides(e.g. cytokines), where such inhibition is provided by application of aθ-defensin and/or θ-defensin analog to a subject in need of such areduction in gene expression. Such a subject can, for example, be inneed of anti-inflammatory treatment. Dosage and dosing frequency of theθ-defensin and/or θ-defensin analog can be selected to provide ameasurable reduction in pro-inflammatory peptide expression in less than10 minutes, less than 15 minutes, less than 20 minutes, less than 30minutes, less than 1 hour, less than 2 hours, less than 3 hours, lessthan 4 hours, less than 6 hours, less than 8 hours, less than 12 hours,less than 16 hours, and/or less than 24 hours from administration of aninitial dose. The reduction in pro-inflammatory peptide expression canpersist for at least 1 hour, at least 2 hours, at least 4 hours, atleast 6 hours, at least 8 hours, at least 12 hours, at least 16 hours,at least 24 hours, at least 48 hours, at least 72 hours, and/or morethan 72 hours from establishment of the reduction of pro-inflammatorypeptide expression effect. Dosing schedules can be selected for dosingintervals ranging from three times a day to once a week. Such areduction in gene expression can result in gene expression levels thatare similar or identical to expression levels of pro-inflammatorypeptides (e.g. cytokines) in the absence of an inflammatory stimulus. Insome embodiments the θ-defensin and/or θ-defensin analog can be providedas a constant infusion. Such a reduction in gene expression can resultin gene expression levels that are similar or identical to expressionlevels of pro-inflammatory peptides (e.g. cytokines) in the absence ofan inflammatory stimulus. An inflammatory stimulus can be an acuteinflammatory condition characterized by sudden and severe onset, andinclude sepsis, traumatic injury, thermal burns, chemical burns,electrical burns, radiation burns, acute allergic reactions, andinhalation injuries. Similarly, an inflammatory stimulus can be achronic inflammatory condition characterized by gradual onset andpersistence over time, and include rheumatoid arthritis, ankylosingspondylitis, inflammatory bowel disease, ulcerative colitis, autisticenterocolitis, psoriasis, psoriatic arthritis, Crohn's disease, Behcet'sdisease, lupus, hidradenitis suppurativa, refractory asthma, colitis,dermatitis, diverticulitis, hepatitis, nephritis, Parkinson's disease,Alzheimer's disease, Huntington's disease, congestive heart disease,atherosclerosis, and uveitis.

Another embodiment of the inventive concept is a composition thatincludes a θ-defensin and/or θ-defensin analog in an amount and formsuitable for reducing expression of one, two, or more than twopro-inflammatory peptide(s) (e.g. cytokines). Such a composition can bein the form of a solid, powder, liquid, gel, and/or suspension. Such acomposition can be formulated to be administered by injection, infusion,orally, rectally, vaginally, as an inhalant (e.g. a mist or powder),and/or topically. The θ-defensin and/or θ-defensin analog can beprovided to provide a dose ranging from 1 μg to 10 mg per kg of bodyweight of an individual to be treated. Such compositions can alsoinclude additional components that do not have direct activity inreducing inflammation and/or expression of a gene encoding apro-inflammatory peptide, such as excipients, flavorants, stabilizers,and/or bulking agents. In some embodiments the composition can includepharmaceutically active compounds that are useful in co-therapy, forexample an antibiotic, antihistamine, bronchodilator, steroid, and/ornon-steroidal anti-inflammatory compound.

Another embodiment of the inventive concept is the use of a θ-defensinand/or θ-defensin analog in the formulation of a medicament useful fortreating an acute and/or chronic inflammatory condition by inhibition ofthe gene expression of one, two, or more than two pro-inflammatorypeptides (e.g. cytokines), where such inhibition is provided byapplication of a θ-defensin and/or θ-defensin analog to a subject inneed of anti-inflammatory treatment. Dosage and dosing frequency of theθ-defensin and/or θ-defensin analog can be selected to provide relieffrom inflammation and/or measurable reduction in pro-inflammatorypeptide expression in less than 10 minutes, less than 15 minutes, lessthan 20 minutes, less than 30 minutes, less than 1 hour, less than 2hours, less than 3 hours, less than 4 hours, less than 6 hours, lessthan 8 hours, less than 12 hours, less than 16 hours, and/or less than24 hours from administration of an initial dose. Relief frominflammation and/or reduction in pro-inflammatory peptide expression canpersist for at least 1 hour, at least 2 hours, at least 4 hours, atleast 6 hours, at least 8 hours, at least 12 hours, at least 16 hours,at least 24 hours, at least 48 hours, at least 72 hours, and/or morethan 72 hours from establishment of the anti-inflammatory or reductionof pro-inflammatory peptide expression effect. Dosing schedules can beselected for dosing intervals ranging from three times a day to once aweek. In some embodiments the θ-defensin and/or θ-defensin analog can beprovided as a constant infusion. Acute inflammatory conditions that canbe treated in this manner are characterized by sudden and severe onset,and include sepsis, traumatic injury, thermal burns, chemical burns,electrical burns, radiation burns, acute allergic reactions, andinhalation injuries. Chronic inflammatory conditions that can be treatedin this manner are characterized by gradual onset and persistence overtime, and include rheumatoid arthritis, ankylosing spondylitis,inflammatory bowel disease, ulcerative colitis, autistic enterocolitis,psoriasis, psoriatic arthritis, Crohn's disease, Behcet's disease,lupus, hidradenitis suppurativa, refractory asthma, colitis, dermatitis,diverticulitis, hepatitis, nephritis, Parkinson's disease, Alzheimer'sdisease, Huntington's disease, congestive heart disease,atherosclerosis, and uveitis.

It should be appreciated that θ-defensins are only found in non-humanprimates. As such, presence of a θ-defensin or θ-defensin analog in ahuman being cannot be considered a naturally-occurring phenomena.

RTD-1 inhibits secretion of several proinflammatory cytokines by humanbuffy coat cells stimulated with E. coli cells as well as with agonistsfor TLR2, 4, 5, and 8 with particularly marked suppression of TNF-α,IL-1α/β, IL-6, IL-8, and CCL3, and CCL4. Consistent with these findings,10 μg/ml of RTD-1, a concentration that the inventors have found iseffective in blocking TNF secretion from stimulated blood cells, wasalso found to be highly effective in blocking secretion of TNF-α, IL-1β,and IL-8 from LPS stimulated THP-1 cells and human monocytes (see FIG.1). RTD-1 (SEQ ID NO. 1) binds inefficiently to LPS; as a result itsimmune regulatory effects are not mediated by neutralization of LPS.Surprisingly, the inventors have found that RTD-1 suppression of thesecytokines, and of CCL3 and CCL4 is due to down regulation of thecorresponding mRNA levels. This indicates that at least oneanti-inflammatory mechanism of θ-defensins is through regulation ofinflammatory gene expression. It should be appreciated that this effectis selective, as RTD-1 (SEQ ID NO. 1) alone does not affect cytokinemRNA or protein levels in unstimulated cells (see FIGS. 1 and 2).

The inventors also analyzed the effect of θ-defensin peptide treatmenton NF-κB activation in THP-1 cells treated with LPS and Pam3CSK4,agonists for TLRs 4, 1/2 (cell surface) respectively, and in HEK BluehTLR9 cells treated with ODN2006, agonist for TLR9 (intracellular).Surprisingly, RTD-1 treatment markedly inhibited NF-κB activation byeach agonist (see FIGS. 3A, 3B, 3C, 3D, and 3E). This inhibitory effectis dose-dependent. RTD-1 was found to inhibit translocation of NF-κB p65to the nucleus in LPS stimulated macrophages (see FIGS. 4A and 4B) andstabilized IκBα (see FIG. 6). Notably, the human α-defensin HNP-1 had noinhibitory activity (see FIG. 3B). It should be appreciated that anacyclic form of RTD-1 (S7; see FIG. 3C) did not inhibit NF-κBactivation, demonstrating that the cyclic structure of this macrocyclicpeptide is essential for this activity.

Previously, inventor(s) showed that the RTD-1-mediated blockade of TNF-αsecretion by E. coli stimulated whole blood was extremely rapid.Inventors found that RTD-1 (SEQ ID NO. 1) reduced TNF-α-mediated NF-κBactivation in THP-1 cells, but to a lesser extent than the peptide'seffect on LPS stimulation (see FIGS. 4A and 4B). Similarly, RTD-1suppressed both TNF-α and IL-1β secretion by LPS-treated THP-1 cells.This is in contrast to the effects of marimastat (see FIG. 5), a potentinhibitor of the TACE/ADAM17 sheddase that generates soluble TNF-α andwhich did not inhibit IL-1β release. Thus blockade of TNF-α release, andsubsequent suppression of autocrine signaling, cannot alone account forthe immunomodulatory effects of RTD-1 (SEQ ID NO. 1).

Inventors also analyzed the effect of RTD-1 (SEQ ID NO. 1) on signalingkinases implicated in LPS-induced inflammatory responses in macrophages.RTD-1 (SEQ ID NO. 1) inhibited LPS-induced phosphorylation of severalinflammatory signaling proteins including p38, JNK, ERK2, CREB and H5P27(see FIGS. 6 and 7). Inhibition of CREB and H5P27 phosphorylation isconsistent with the suppression of p38 MAP kinase which phosphorylatesthese proteins (see FIG. 10). The p38 MAP kinase is implicated instabilization of mRNAs containing AU-rich elements of immune responsegenes such as TNF-α, IL-6, CCL3, IL-8, and other immune response genes.The MEK-ERK pathway is involved in nucleocytoplasmic transport of TNF-αmRNA in mouse macrophage cells. Stimulation of JNK MAP kinase leads toactivation of the AP-1 class of transcription factors which are involvedin activation of immune response genes. The inventors have found that,surprisingly, RTD-1 (SEQ ID NO. 1) regulates inflammatory signaling byinhibiting the phosphorylation of several inflammatory signalingproteins, for example signaling proteins involved in these pathways.

THP-1 cell exposure to RTD-1 alone was found to result inphosphorylation of Akt (see FIG. 7), which occurs very rapidly afterRTD-1 treatment and produces a sustained effect (see FIGS. 8A, 8B, and8C). Surprisingly, the inventors found that this effect is highlyselective as RTD-1 did not increase baseline phosphorylation of otherkinase targets evaluated in otherwise untreated cells (see FIGS. 7, 8A,8B, and 8C). This effect is also found in other cell types, as RTD-1(SEQ ID NO. 1) inhibited the degradation of IκBα, phosphorylation of p38MAP kinase in LPS-stimulated human monocytes and also stimulated Aktphosphorylation in these cells (see FIG. 9).

This RTD-1 (SEQ ID NO. 1)-induced phosphorylation of Akt was blocked bya specific PI3K inhibitor, indicating that the PI3K-Akt pathway can beinvolved in mediating the anti-inflammatory effects of RTD-1 (SEQ IDNO. 1) (see FIGS. 8A, 8B, and 8C). PI3K-Akt is a negative regulator ofNF-κB and MAP kinase pathways and inhibits downstream TNF-α geneexpression in LPS stimulated cells. Inhibition of the PI3K-Akt pathwayincreases the mortality and circulating pro-inflammatory cytokine levelsin endotoxemic mice as well as in mice with polymicrobial sepsis inducedby cecal ligation and puncture (CLP). This suggests that a θ-defensinand/or θ-defensin analog can act through the PI3K-Akt pathway to reduceor eliminate release of pro-inflammatory peptides (such aspro-inflammatory cytokines), thereby reducing or eliminating anundesirable inflammatory response in a subject treated with such aθ-defensin and/or θ-defensin analog. Interestingly both human α-defensin(e.g. HNP1) and human β-defensin (e.g. HBD2 and HBD3) can stimulateexpression and secretion of pro-inflammatory cytokines from humanconjunctival epithelial cells, which is accompanied by phosphorylationof Akt. Both α- and β-defensins are known to regulate cellular responsesthrough receptor mediated pathways. The human β-defensins, HBD2 andHBD3, induce chemotaxis in monocytes through the CCR2 receptor and HBD2induces chemotaxis in dendritic and T cells through the CCR6 receptors.To date there have been no reports of a receptor for θ-defensins,suggesting that θ-defensin and/or θ-defensin analogs can utilize eitherheretofore undiscovered receptors for these non-human peptides or act ina receptor-independent fashion. Studies are ongoing to identify RTD-1(SEQ ID NO. 1) targets upstream of the PI3K-Akt pathway and therelationship of RTD-1-induced Akt phosphorylation to downstreamsignaling pathways. It should be noted that the suppression of TNF-αsecretion by RTD-1 (SEQ ID NO. 1) is extremely rapid suggesting thatRTD-1 (SEQ ID NO. 1) can also regulate pathways distinct from thosecurrently known to be involved in signaling and expression ofpro-inflammatory genes.

Human β-defensin (hBD-3) inhibits LPS-induced gene transcription andpro-inflammatory cytokine secretion in mouse RAW264.7 macrophages viainhibition of the NF-κB pathway involving signaling through MyD88 andTRIF. It has been theorized that the contrasting pro- andanti-inflammatory properties reported for β-defensins may reflectdifferences in structural features and/or purity of different peptidepreparations. Another possibility is that the proinflammatory versusanti-inflammatory properties of a single peptide can be a function oflocal concentration of the peptide. Without wishing to be bound bytheory, the inventors theorize that θ-defensins, which are expressed atvery high levels in granules of circulating neutrophils and monocytes,provide a physiologic dampening mechanism for inflammatory processesfollowing their release and/or secretion, systemically and/or locally.As such θ-defensins and θ-defensin analogs can act as a safe andeffective anti-inflammatory compound that can be applied to a widevariety of inflammatory conditions, both chronic and acute, and bothsystemic and localized.

In summary, RTD-1 (SEQ ID NO. 1) suppresses NF-κB activation and hencepro-inflammatory peptide (e.g. pro-inflammatory cytokine) expression andrelease. Such immunomodulation occurs at both the TLR and TNF pathways.Evidence for a role for induction of Akt phosphorylation is suggested byRTD-1 (SEQ ID NO. 1) induced P-Akt in both naïve and LPS-stimulatedcells. The immunomodulatory properties of the macrocyclic peptide aredistinct from those of human α-defensins, and RTD-1 (SEQ ID NO. 1)suppresses inflammation by mechanisms that are distinct from those ofhBD-3. Further, since RTD-1, related θ-defensins, and θ-defensindifferentially inhibit TNF secretion from stimulated blood cells,θ-defensin isoforms and θ-defensin analogs can discriminate betweenregulatory pathways that determine whether the subject response toinflammatory stimuli results in health or disease, and therefore canselectively provide anti-inflammatory effects to diseased and/or damagedtissue.

Examples Reagents

Phorbol 12-myristate 13-acetate (PMA), marimastat, and phenyl methylsulfonyl fluoride (PMSF) were from Sigma Chemical Co. (St. Louis, Mo.),E. coli K12 lipopolysaccharide (LPS), Pam3CSK4, ODN2006 and Quantibluewere from Invivogen (San Diego, Calif.). Anti-phospho-p38 MAP kinase,anti-IκBα, anti-phospho-SAPK/JNK, anti-NF-κB (p65), anti-phospho-Akt(Ser473) and anti-Akt pan antibodies were from Cell Signaling Technology(Danvers, Mass.). Marimastat and Ly294002 (Cell Signaling Technology)solutions were prepared at 1000× in DMSO. Carrier free TNF-α (CellSignaling Technology) was suspended at 2 μg/ml in phosphate bufferedsaline (PBS) containing 5% heat inactivated (HI) fetal bovine serum(FBS). Synthetic RTD-1 (SEQ ID NO. 1) hydrochloride (>99%) was preparedas described in Tang et al, Science 286:498-502 (1999). Human α-defensinHNP-1 was purified from neutrophils. Stock solutions of RTD-1 (SEQ IDNO. 1), S7 peptide and HNP-1 were prepared in sterile 0.01% acetic acid.It should be appreciated that the S7 peptide is a linearized form ofRTD-1 (SEQ ID NO. 1), and thus shares the same amino acid sequence whilehaving an acyclic structure.

Cell Culture:

THP-1 monocytes (ATCC, Manassas, Va.) were cultured in RPMI 1640+10% FBSand penicillin/streptomycin (P/S). THP-1 Dual cells (Invivogen, SanDiego, Calif.) which express the NF-κB reporter, secreted embryonicalkaline phosphatase (SEAP), were grown in RPMI 1640+10% HI-FBS andantibiotics. THP-1 cell differentiation was induced by treatment ofcells (˜3.3×10⁵ cells/ml) with 100 nM PMA and cultured for 2 days.HEK-Blue™ hTLR9 cells (Invivogen) were grown in DMEM medium containing10% HI-FBS, P/S and normocin. Cells were cultured at 37° C. in 5% CO₂.Cytokine assays. PMA-differentiated THP-1 cells (˜8×10⁵ cells) culturedin 6-well plates were washed twice with complete medium and grown infresh medium containing 1% FBS for 24 hours. Cells were washed twicewith fresh medium and incubated for 2 hours prior to furthermanipulation. Cells were stimulated with 100 ng/ml LPS in the presenceor absence of 10 μg/ml RTD-1 or vehicle (0.01% acetic acid) for 2 hours.In some experiments, cells were first incubated for 1 hour with 10 μg/mlof marimastat or vehicle (DMSO), after which LPS and/or RTD-1 were addedand incubated for 2 hours as above. Peripheral blood CD14+ monocytes(Lonza, Walkersville, Md.) were thawed, rinsed with RPMI 1640 containing10% HI FBS, P/S and normocin and then resuspended in RPMI 1640containing 1% HI FBS plus antibiotics as above (10⁶ cells/nil) for 2hours. The cells were then stimulated for 4 hours as described in thelegend. Medium was collected, clarified by centrifugation first at 250×gfor 8 minutes and then at 5000×g for 5 minutes. TNF-α, IL-1β and IL-8were quantified by ELISA (Life Technologies, Carlsbad, Calif.).

Real Time Quantitative PCR.

Differentiated THP-1 cells were treated with LPS and RTD-1 for 2 hoursas above, washed with PBS and RNA was isolated using an RNA mini kit(Zymo Research, Irvine, Calif.) or with RNeasy mini kit (Qiagen,Valencia, Calif.). RNA integrity was confirmed on agarose gels and RNAsamples with A260/280 and A260/230 ratios ≧1.7 were used to generatecDNA. Briefly, 400 ng RNA was incubated with genomic DNA (gDNA)elimination (GE) buffer for 5 minutes at 4° C. and reverse transcribedusing the RT² first strand synthesis kit (Qiagen). Custom PCR arrays (SABiosciences, Frederick, Md.) containing human genes of interest andcontrols (for reverse transcriptase and PCR efficiency, RTC and PPCrespectively) were used for simultaneous real time PCR analysis. Mastermix containing cDNA and RT² SYBR Green™ was added to array wells. PCRcycling parameters were: 1 cycle 10 minutes at 95° C., 40 cycles of 15seconds at 95° C., 1 minute at 60° C. on a BioRad C1000 Thermal Cyclerequipped with a CFX96 real time system. Melting curve analysis confirmeda single amplicon with CT PPC=20±2 and ACT (RTC-PPC)<5 for all samples.The ACTB gene was used for normalization of gene expression and foldstimulation (compared to untreated cells) was calculated using the ΔΔCTmethod. Absence of gDNA in cDNA samples (CT>35) was confirmed using ahuman gDNA primer (SA Biosciences).

NF-κB Activity Assay.

One ml aliquots of PMA-differentiated THP-1 Dual cells (3.3×10⁵cells/nil) in 24 well tissue culture plates were starved for 1 day incomplete medium with 1% HI FBS as described above. For experiments withS7 peptide, 0.5 ml cultures in 48 well plates were used. Fresh mediumcontaining 1% HI FBS was added to the cells and after 2 hours cells werestimulated with 100 ng/ml LPS or 25 ng/ml Pam3CSK4 in the presence ofRTD-1 or 0.01% acetic acid (HOAc, vehicle). In some experiments cellswere pre-incubated with marimastat, Ly294002 or vehicle for 1 hour priorto addition of LPS and/or RTD-1 (SEQ ID NO. 1). Following overnightincubation, medium was clarified by centrifugation and SEAP activity wasdetermined using Quantiblue reagent at 37° C. and measurement ofabsorbance at 625 nm. To test the effect of RTD-1 on TLR9-dependentNF-κB activation, 2×10⁵ HEK-Blue™ hTLR9 cells/ml were cultured for 1 dayin 24 well plates and stimulated overnight with ODN2006 with or withoutRTD-1. Medium was harvested and SEAP activity was assayed usingQuantiblue™ as above. NF-κB activation was defined as fold stimulationof SEAP activity with respect to control cells.

NF-κB DNA-Binding Activity.

Nuclear extracts were prepared (nuclear extraction kit; Cayman ChemicalCo., Ann Arbor, Mich.) from differentiated THP-1 cells treated for 30minutes with vehicle, LPS (100 ng/ml) or TNF-α (2 ng/ml) in the presenceor absence of 10 μg/ml RTD-1 (SEQ ID NO. 1). NF-κB DNA binding wasquantified by p65 ELISA (Cayman Chemical Co.). Non-specific DNA-binding,positive control extract and positive control extract treated withcompetitor DNA control included with the ELISA were used to confirmspecificity of DNA-binding.

Isolation of Monocytes from Blood.

EDTA-anticoagulated blood was incubated with human monocyte enrichmentcocktail (Stem Cell Technologies, Vancouver, BC, Canada) for 20 minutesand layered on Ficoll Paque™ in a Sepmate™ 50 tube (Stem Cell Tech). Thecells were centrifuged at 1200×g for 10 minutes and the monocytecontaining layer of cells was isolated, washed with PBS containing 2% HIFBS and 1 mM EDTA.

Immunoblot Analyses.

Differentiated THP-1 cells (˜8×10⁵ cells/2.5 ml medium) were starved incomplete medium containing 1 FBS for 1 day as above, and fresh 2.5 mlRPMI 1640+1% FBS+P/S was added for 2 hours. For experiments with freshmonocytes, cells (>80% CD14+) were resuspended (˜10⁶ cells/nil) inmonocyte attachment medium (Promocell, Heidelberg, Germany) in wells ofa 12 well plate for 2 hours at 37° C. in 5% CO₂. The cells were thenwashed two times with RPMI medium containing 1% HI FBS and suspendedovernight in RPMI medium containing 1% HI FBS. Cells were stimulatedwith agonists with or without RTD-1 (SEQ ID NO. 1) as described in thefigure legends after which cells were washed with PBS and extracts wereprepared in cell lysis buffer+1 mM PMSF. Protein content was determinedusing the BCA method (BioRad, San Diego, Calif.) and extracts resolvedon SDS-tricine gels and transferred to nitrocellulose membrane. Themembranes were probed with the indicated antibodies and developed usinganti-mouse or anti-rabbit HRP conjugated secondary antibodies anddetected by chemiluminescence. A phosphoprotein antibody array(Phospho-MAPK™; R&D systems Minneapolis, Minn.) was used to analyzeactivation of phosphoprotein kinases. Differentiated THP-1 cells(1.6×10⁶ cells) were treated with 100 ng/ml LPS, 10 μg/ml RTD-1 (SEQ IDNO. 1), 100 ng/ml LPS+10 μg/ml RTD-1, or vehicle for 30 minutes. Arrayswere probed with 200 μg of cell extract protein and chemiluminescence ofspots from arrays or bands from western blotting was quantified usingNIH ImageJ software, background subtracted, normalized and plotted.

Statistical Analysis.

Standard deviation (SD) and standard error of mean (SEM) were calculatedfor experiments repeated 3 and 4 times respectively and Student's t-testwas performed to evaluate the significance of peptide effects asindicated in the figure legends, difference was considered significantif P<0.05. Microsoft Excel was used for all statistical analyses.

RTD-1 (SEQ ID NO. 1) Suppression of Expression and Release ofPro-Inflammatory Cytokines.

Inventor(s) have found that RTD-1 (SEQ ID NO. 1) suppressed secretion ofseveral inflammatory cytokines by peripheral blood leukocytes stimulatedby agonists of TLRs 2, 4, 5, and 8 and the inhibition of TNF-α, IL-1β,and IL-8 release was most notable. RTD-1 (SEQ ID NO. 1) treatmentmarkedly (>90%) suppressed secretion of TNF-α, IL-1β and IL-8 byLPS-stimulated primary human monocytes and THP-1 macrophages (see FIG.1). RTD-1 (SEQ ID NO. 1) alone had no effect on THP-1 cytokine secretion(see FIG. 1). The cytokine inhibitory effects in THP-1 macrophagesclosely resemble the responses obtained with human blood buffy coatcells and human monocytes indicating that THP-1 cells provide anappropriate model for exploring RTD-1 (SEQ ID NO. 1)-mediatedanti-inflammatory mechanisms.

In experiments similar to those described in FIG. 1, inventors analyzedthe effect of RTD-1 (SEQ ID NO. 1) on cytokine mRNA expression byLPS-stimulated THP-1 cells, focusing on TNF-α, IL-1β, IL-8, CCL3, andCCL4. As shown in FIG. 2, RTD-1 (SEQ ID NO. 1) markedly inhibitedLPS-induced mRNA expression of each of the 5 cytokines. The addition ofRTD-1 (SEQ ID NO. 1) had no effect on the expression of GAPDH and thepeptide alone had no effect on cytokine mRNA expression. These results(see FIGS. 1 and 2) demonstrate potent regulation of pro-inflammatorycytokine expression and release by RTD-1 (SEQ ID NO. 1) in LPSstimulated macrophages.

Regulation of NF-κB Pathway by RTD-1 (SEQ ID NO. 1).

Given the central role of NF-κB signaling in inflammation, inventorstheorized that RTD-1 (SEQ ID NO. 1) modulates this pathway in THP-1cells stimulated with different TLR agonists. LPS (TLR4 agonist)treatment of THP-1 cells induced a ˜7-fold activation of NF-κB (FIG.3A). Simultaneous treatment with RTD-1 (SEQ ID NO. 1) and LPS suppressedNF-κB activation by ˜70%, whereas RTD-1 (SEQ ID NO. 1) alone had noeffect (FIG. 3A). In addition, RTD-1 had no direct effect on theenzymatic activity of the SEAP reporter (LPS/RTD-1 (FIG. 3A); inhibitionof NF-κB activation by RTD-1 (SEQ ID NO. 1) was dose dependent (FIG.3B). Notably, no inhibition of NF-κB activity was observed whenequimolar concentrations of human neutrophil α-defensin HNP-1 (FIG. 3B)or an acyclic form of RTD-1 (SEQ ID NO. 1) (S7; FIG. 3C) were used inplace of RTD-1 (SEQ ID NO. 1).

To characterize the effects of RTD-1 on NF-κB activity induced by otherstimuli, inventors analyzed responses of THP-1 cells stimulated with theTLR1/2 agonist Pam3CSK4 and HEK-Blue hTLR9 cells stimulated with TLR9agonist ODN2006 with and without addition of RTD-1 (SEQ ID NO. 1). Bothligands stimulated NF-κB activation, 5- and 6-fold respectively.Addition of 10 μg/ml of RTD-1 inhibited Pam3CSK4-induced NF-κB activityby ˜70%, and the peptide suppressed ODN2006-mediated activation tobaseline levels (FIGS. 3D and 3E, respectively).

Inventors further characterized the effect of RTD-1 (SEQ ID NO. 1) onLPS-stimulation of THP-1 cells by analyzing the effect of the peptide onDNA binding by NF-κB. LPS treatment induced a 4-fold increase in NF-κBDNA-binding (FIG. 4A). However, co-incubation of LPS and RTD-1 (SEQ IDNO. 1) reduced NF-κB DNA binding to baseline levels (FIG. 4A) whichcorrelated with the levels of NF-κB p65 subunit levels in nuclearextracts from cells treated with LPS+/−RTD-1 (SEQ ID NO. 1) as detectedby western blotting (FIG. 4A).

Inventors also tested the effect of RTD-1 (SEQ ID NO. 1) on TNF-αstimulation of NF-κB DNA binding. Compared with TNF-α alone, RTD-1 (SEQID NO. 1) suppressed NF-κB DNA binding by ˜35%, substantially lessinhibition than observed in LPS-stimulated cells. The decrease in RTD-1(SEQ ID NO. 1) inhibition of TNF-α-stimulated NF-κB DNA bindingcorrelated with the levels of p65 in nuclear extracts of treated anduntreated THP-1 cells (FIG. 4B).

The anti-inflammatory effects of RTD-1 (SEQ ID NO. 1) were compared tothose of marimastat, a potent inhibitor of TNF-α converting enzyme(TACE/ADAM17), the sheddase that produces soluble TNF-α from themembrane bound precursor. Marimastat efficiently blocked release ofTNF-α by LPS-stimulated THP-1 cells, but did not affect secretion ofIL-1β (FIG. 5). This result is in contrast to the broader inhibitoryactivity of RTD-1 (SEQ ID NO. 1), which blocked both cytokineseffectively (FIGS. 1 and 5A). Notably, marimastat treatment had noeffect on LPS-stimulated NF-κB activation (FIG. 5B), in contrast toRTD-1 (SEQ ID NO. 1) which was a potent inhibitor of this activity inthe presence or absence of marimastat (FIG. 5B).

Signaling Pathways Affected by RTD-1 (SEQ ID NO. 1).

LPS binding to TLR4 initiates a cascade of signaling events that leadsto activation of a complex containing TGF-β-activated kinase 1 (TAK1)which phosphorylates IκB kinase (IKK). Activated IKK phosphorylates theNF-κB inhibitor IκBα triggering its degradation which activates NF-κBfor nuclear translocation (FIG. 6). LPS-induced activation of TAK1 alsostimulates numerous kinase pathways. RTD-1 (SEQ ID NO. 1) was effectivein blocking the degradation of IκBα in LPS stimulated macrophages andthis was accompanied by inhibition of p38 MAP kinase and JNK1/2 kinasephosphorylation. RTD-1 (SEQ ID NO. 1) effects when used alone wereselective, showing no effect on IκBα levels or on phosphorylation of p38MAP and JNK1/2 kinases (FIG. 6).

Inventors used phosphoprotein antibody arrays to identify otherpotential signaling targets of RTD-1 (SEQ ID NO. 1). Extracts ofLPS-stimulated macrophages contained elevated levels of phosphorylatedCREB, ERK2, H5P27, JNK2, p38α and p38γ compared to control cells (FIG.7). RTD-1 (SEQ ID NO. 1) inhibited phosphorylation of each of theseproteins to baseline levels (FIG. 7, lower panel) in such stimulatedcells, however treatment with RTD-1 (SEQ ID NO. 1) alone did notsuppress the phosphorylation of these proteins (FIG. 7).

In contrast, RTD-1 (SEQ ID NO. 1) alone or in combination with LPSselectively stimulated phosphorylation of Akt1 (FIG. 7). The kinetics ofRTD-1 (SEQ ID NO. 1) effects on Akt expression and phosphorylation weredetermined by treating THP-1 cells with RTD-1 (SEQ ID NO. 1) alone orvehicle and analyzing Akt and P-Akt levels. RTD-1 (SEQ ID NO. 1)treatment induced a 6.5 fold increase in P-Akt within 10 minutes ofpeptide treatment and P-Akt levels remained elevated by ˜2.5 fold for atleast 4 hours (FIG. 8A). Additional experiments were performed toidentify the role of PI3K in the observed RTD-1-dependent stimulation ofAkt phosphorylation. Addition of PI3K inhibitor Ly294002 markedlyreduced RTD-1 (SEQ ID NO. 1) induced P-Akt levels (FIG. 8B)demonstrating that RTD-1 effect was upstream of Akt. Because thePI3K-Akt pathway negatively regulates activation of NF-κB and MAP kinasepathways, inventors characterized the effect of Ly294002 on the RTD-1mediated inhibition of NF-κB activation in LPS stimulated THP-1 Dualmacrophage cells. Ly294002 reversed the suppression of NF-κB activationby RTD-1 (SEQ ID NO. 1) in dose-dependent manner (FIG. 8C), evidencethat stimulation of Akt phosphorylation contributes to the observedanti-inflammatory effects of RTD-1 (SEQ ID NO. 1).

It should be appreciated that the effects of θ-defensin and/orθ-defensin show a high degree of selectivity, exerting effectsselectively to cells exposed to inflammatory stimuli and specificallyimpacting on certain processes and/or pathways in such cells. As such,treatment with θ-defensin and/or θ-defensin can provide selectivetherapy, particularly in immune-compromised or potentiallyimmune-compromised individuals. Such therapy can effectively reduceexpression of pro-inflammatory peptides in an individual while notinducing an immunocompromised state (i.e. not producing animmunocompromised state in a non-immunocompromised individual and/or notexacerbating the degree of immunocompromise in an immunocompromisedindividual).

Inventors also characterized the effects of RTD-1 (SEQ ID NO. 1) onsignaling pathways in primary human monocytes. RTD-1 (SEQ ID NO. 1)alone did not induce degradation of IκBα but it reduced the degradationof IκBα in LPS stimulated monocytes (FIG. 9). RTD-1 (SEQ ID NO. 1)inhibited p38 MAP kinase phosphorylation in LPS stimulated monocytes,but the peptide alone had no effect on p38 MAP kinase phosphorylation(FIG. 9). However, as observed in experiments with THP-1 macrophages,increased phosphorylation of Akt was observed in monocytes treated withLPS, LPS and RTD-1 (SEQ ID NO. 1) or with RTD-1 (SEQ ID NO. 1) alonecompared to control cells (FIG. 9). Thus, RTD-1 (SEQ ID NO. 1) regulatesNF-κB, MAP kinase and PI3K-Akt signaling pathways across a range of celltypes, including human blood monocytes and THP-1 cells.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refer to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

REFERENCES

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Where a definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

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1. A method for modulation of multiple pro-inflammatory peptides in ahuman or nonhuman mammal, comprising administering a θ-defensin orθ-defensin analog to a subject in need thereof, in an amount effectiveto modulate a plurality of pro-inflammatory peptides by suppressing geneexpression of the pro-inflammatory peptides.
 2. The method of claim 1,wherein the θ-defensin is selected from the group consisting of RTD-1(SEQ ID NO. 1), RTD-2 (SEQ ID NO. 5), RTD-3 (SEQ ID NO. 6), RTD-4 (SEQID NO. 7), RTD-5 (SEQ ID NO. 8), and RTD-6 (SEQ ID NO. 9).
 3. The methodof claim 1, wherein the θ-defensin analog is selected from the groupconsisting of RTD 1-27 (SEQ ID NO. 2), RTD 1-28 (SEQ ID NO. 3) and RTD1-29 (SEQ ID NO. 4).
 4. The method of claim 1 wherein the modulation ofinjurious cytokines or chemokines is mediated by suppression NF-κBsignaling by the macrocyclic peptide.
 5. The method of claim 1 whereinthe modulation of the pro-inflammatory peptides is mediated bysuppression of MAP kinase activation by the θ-defensin or θ-defensinanalog.
 6. The method of claim 1 wherein the modulation of thepro-inflammatory peptides is a result of activation of the PI3K/Aktpathway by the θ-defensin or θ-defensin analog.
 7. The method of claim 1wherein modulation of the pro-inflammatory peptides is a result ofsuppression of an immune response induced by interaction between aToll-like receptor agonist and a receptor for the Toll-like receptoragonist.
 8. The method of claim 1 wherein the θ-defensin or θ-defensinanalog interrupts autocrine or paracrine proinflammatory signaling by acytokines or a chemokine.
 9. The method of claim 1 wherein theθ-defensin or θ-defensin analog suppresses a proinflammatory MAP kinase.10. The method of claim 9, wherein the proinflammatory MAP kinase isselected from the group consisting of JNK, p38 MAP kinase, and ERK. 11.The method of claim 1 wherein the θ-defensin or θ-defensin analogsuppresses proinflammatory effects of the NF-κB pathway by inducingphosphorylation of Akt.
 12. A method for suppressing a pluralitypro-inflammatory peptides without producing an immunocompromised state,comprising administering a θ-defensin or θ-defensin analog, to a humanor nonhuman subject in need thereof, in an amount effective to suppressthe plurality of injurious cytokines or chemokines and that is alsoineffective in inducing the immunocompromised state.
 13. A method forsuppressing a pro-inflammatory peptide without producing animmunocompromised state in the setting of infection comprisingadministering a θ-defensin or θ-defensin analog, to an infected human ornonhuman subject in need thereof, in an amount effective to suppress theplurality of injurious cytokines or chemokines and that is alsoineffective in inducing the immunocompromised state.
 14. A method forsuppressing a plurality of pro-inflammatory peptides without producingan immunocompromised state in a setting of immunosuppression, comprisingadministering a θ-defensin or θ-defensin analog, to an immunosuppressedhuman or nonhuman subject in need thereof, in an amount effective tosuppress the plurality of injurious cytokines or chemokines and that isalso ineffective in inducing the immunocompromised state.
 15. A methodfor suppressing a plurality of pro-inflammatory peptides withoutproducing an immunocompromised state in a setting of autoimmune disease,comprising administering a θ-defensin or θ-defensin analog, to a humanor nonhuman subject in need thereof and having an autoimmune disease, inan amount effective to suppress the plurality of injurious cytokines orchemokines and that is also ineffective in inducing theimmunocompromised state.
 16. The methods of any one of claims 12 to 15,wherein at least one of the plurality of pro-inflammatory peptides isselected from the group consisting of tumor necrosis factor alpha(TNFα), IL-1β, IL-6, IL-8, CCL3, and CCL4.
 17. A composition formodulation of multiple pro-inflammatory peptides in a human or nonhumanmammal, comprising a θ-defensin or θ-defensin analog to a subject inneed thereof, in an amount effective to modulate a plurality ofpro-inflammatory peptides by suppressing gene expression of thepro-inflammatory peptides.
 18. The composition of claim 17, wherein theθ-defensin is selected from the group consisting of RTD-1 (SEQ ID NO.1), RTD-2 (SEQ ID NO. 5), RTD-3 (SEQ ID NO. 6), RTD-4 (SEQ ID NO. 7),RTD-5 (SEQ ID NO. 8), and RTD-6 (SEQ ID NO. 9).
 19. The composition ofclaim 17, wherein the θ-defensin analog is selected from the groupconsisting of RTD 1-27 (SEQ ID NO. 2), RTD 1-28 (SEQ ID NO. 3) and RTD1-29 (SEQ ID NO. 4).
 20. The composition of claim 17 wherein themodulation of injurious cytokines or chemokines is mediated bysuppression NF-κB signaling by the macrocyclic peptide.
 21. Thecomposition of claim 17 wherein the modulation of the pro-inflammatorypeptides is mediated by suppression of MAP kinase activation by theθ-defensin or θ-defensin analog.
 22. The composition of claim 17 whereinthe modulation of the pro-inflammatory peptides is a result ofactivation of the PI3K/Akt pathway by the θ-defensin or θ-defensinanalog.
 23. The composition of claim 17 wherein modulation of thepro-inflammatory peptides is a result of suppression of an immuneresponse induced by interaction between a Toll-like receptor agonist anda receptor for the Toll-like receptor agonist.
 24. The composition ofclaim 17 wherein the θ-defensin or θ-defensin analog interruptsautocrine or paracrine proinflammatory signaling by a cytokines or achemokine.
 25. The composition of claim 17 wherein the θ-defensin orθ-defensin analog suppresses a proinflammatory MAP kinase.
 26. Thecomposition of claim 25, wherein the proinflammatory MAP kinase isselected from the group consisting of JNK, p38 MAP kinase, and ERK. 27.The composition of claim 17 wherein the θ-defensin or θ-defensin analogsuppresses proinflammatory effects of the NF-κB pathway by inducingphosphorylation of Akt.